阿托伐他汀对大鼠主动脉增龄性内皮功能障碍的干预研究
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摘要
人口老龄化是指总人口中年轻人口数量减少、年长人口数量增加而导致的老年人口比例相应增长的动态,是20世纪下半期到21世纪初出现的一个世界性问题。目前世界老龄人口,即60岁及以上的人口达到7.5亿。到2050年,预计老龄人口将达到20亿,并且80%的老年人将生活在发展中国家。2013年,我国60岁以上老年人口已达到2.02亿,占全国总人口的14.8%；预计到2015年和2020年,这一比例将分别达到15%和18%。通常,65岁以上的人口比率超过总人口的7%,就被称为“老龄化社会”,而超过了14%就被称为“老龄社会”。世界上所有发达国家都已经进入老龄社会,许多发展中国家正在或即将进入老龄社会。中国在1999年已进入“老龄社会”,随之而来的问题是老年人的健康状况不容乐观,老年人对于医疗、保健、护理以及生活服务的需求大大超过其他人。因此倡导健康老龄化,促进老龄人口的健康是医疗工作者和社会的使命。理论上抗衰老治疗可以改变衰老引起的生化和分子学方面的变化,纠正衰老相关的生理学变化,减少对衰老相关疾病的易感性。美国抗衰老医学学会(American Academy of Anti-Aging Medicine, A4M)宣称：“衰老相关的能力丧失由机体的生理功能障碍引起,许多情况下这些功能障碍可被内科治疗所改善。人类的寿命可望延长,老年人的生活质量可望改善”。人类的衰老是可以被解释并且可能被延缓的,寻找延缓衰老的分子靶点并加以干预是当前研究热点。
因为全身各器官组织的正常工作有赖于循环系统的正常供血,而血管的正常功能在维持循环系统的正常运行中发挥着不可或缺的作用,所以衰老过程中血管功能的改变将极大地影响着人体整体的机能。随着机体的衰老,血管也相应地衰老,表现为血管结构和功能的改变。增龄性血管功能障碍与内皮功能障碍密切相关,表现为阻力和传导动脉的内皮依赖性血管舒张能力进行性下降。因为血管内皮功能障碍是一个系统性的功能紊乱,并且是动脉粥样硬化、冠心病和高血压病的一个关键因素,因此改善内皮功能障碍能够减少心脑血管疾病的发病率和死亡率。虽然离体实验已经证实抑制血管细胞衰老能够改善细胞功能障碍,但是在体的研究仍然需要进一步探讨。
他汀作为3-羟基-3-甲基戊二酸辅酶A还原酶(3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase)抑制剂,除具有调脂作用外,还可以改善血管内皮功能障碍,从而防治一系列的血管相关疾病,如动脉粥样硬化、冠心病和高血压等。尽管如此,之前的研究主要是离体实验,或者短期的他汀干预急性病理血管功能障碍模型。考虑到衰老过程中缓慢而微小的病理变化需要长期的累积才能造成明显的功能改变,建立一个自然衰老动物模型将能够更好地研究他汀对于增龄性血管功能障碍的影响。
几个信号通路已经被证实参与血管衰老以及他汀的抑制效应。一氧化氮(NO)被公认为调节血管功能的关键信号分子。NO由一氧化氮合成酶(Nitric oxide synthase, NOS)合成,而一氧化氮合成酶有三种同分异构体,分别为内皮型一氧化氮合成酶(endothelial Nitric oxide synthase, eNOS)、诱导型一氧化氮合成酶(inducible Nitric oxide synthase, iNOS)和神经型一氧化氮合成酶(neuronal Nitric oxide synthase, nNOS)。NO对于血管功能的影响取决于它所处的环境,包括NO的来源和数量。eNOS以及eNOS来源的NO是钙依赖性的,并发挥有益的作用,但是随着衰老而下降。iNOS以及iNOS来源的NO是非钙依赖性的,并发挥有害的作用,但是随着衰老而上升。生理情况下eNOS合成的NO使血管舒张,从而维持正常血管功能。在病理情况下,iNOS被激活而产生大量NO。一方面,过量NO与活性氧簇(Reactive oxygen species, ROS)相互作用,形成亚硝酸盐(Peroxynitrite, ONOO-),进而诱导脂质过氧化而加速血管衰老,最终导致内皮功能障碍。另一方面,iNOS来源的NO激活精氨酸酶I,使eNOS底物降解,导致eNOS的失偶联而造成血管功能障碍。衰老过程中,主动脉中的iNOS被激活或上调,但是对于iNOS的确切位置和来源依然值得研究。他汀对于NOS同分异构体的作用也存在争议,特别是对于iNOS的影响。因为自然衰老动物模型需要长期的干预和观察,他汀对于自然衰老动物模型内皮功能障碍的研究甚少,需要进一步研究。
SIRT1(Sirtuin-1)是一个烟酰胺腺嘌呤二核苷酸(NAD+)依赖的组蛋白,能够调节内皮细胞衰老和血管功能。SIRT1也能够与eNOS相互作用,并刺激eNOS的表达从而抑制血管细胞衰老和改善内皮功能障碍。虽然离体实验证实,阿托伐他汀可以通过诱导SIRT1抑制细胞衰老,但是阿托伐他汀是否可以同样通过诱导SIRT1而抑制血管老化仍值得探讨。
基于以上观点和研究现状,我们假设衰老过程中血管将出现功能障碍,阿托伐他汀的长期干预治疗可以改善血管功能障碍,并且是通过调节增龄性变化过程的氧化应激损伤以及NOS系统的失衡的结果。同时,基于SIRT1在血管功能中发挥的作用,我们假设SIRT1也能够与NOS系统相互作用,而参与血管功能的改变以及他汀的干预效应。本课题组前期研究显示,不同年龄段的Wistar大鼠通过长期饲养,血管出现一系列的病理变化,包括氧化应激损伤和eNOS的下调。这些增龄性变化提示大鼠血管发生了衰老,因此该模型可作为良好的自然衰老动物模型。本研究采用改进的动物模型,采用各个年龄段雄性Wistar大鼠长期喂养,作为自然衰老模型。本研究围绕以下三个方面进行,不但可以回答上述提出的假设问题,同时可以探讨阿托伐他汀改善内皮功能障碍的作用是否通过其经典的NOS系统,以及对于SIRT1的影响。
一、大鼠血管增龄性内皮功能障碍以及阿托伐他汀的干预作用
本研究以目前公认的反映血管功能的两项指标：内皮依赖性的血管功能检查(乙酰胆碱诱导的血管舒张能力检测)以及非内皮依赖性的血管功能检查(硝普钠诱导的血管舒张能力检测)为主要观察指标。实验分4组：青年对照组(2月龄雄性Wistar大鼠,n=8),中年对照组(4月龄雄性Wistar大鼠喂养基础饲料,饲养至12月龄,n=8),老年对照组(12月龄雄性Wistar大鼠喂养基础饲料,饲养至20月龄,n=8,饲养至19月龄时,死亡1只),阿托伐他汀干预组(12月龄雄性Wistar大鼠喂养添加阿托伐他汀(5mg/kg·d)的饲料,饲养至20月龄,n=8)。饲养8个月后,开胸取胸主动脉,测定上述指标。结果显示：青年组、中年对照组和老年对照组之间的比较发现,内皮依赖性的血管舒张功能随着年龄增长而下降(P<0.05或者0.01),非内皮依赖性的血管舒张功能随着年龄增长无变化(P>0.05)。这提示增龄导致了大鼠血管内皮功能障碍。
此外,长期的阿托伐他汀干预对血管功能影响显示：与老年对照组相比,长期的阿托伐他汀干预后,内皮依赖性舒张能力得到了提高(P<0.05或者0.01),非内皮依赖性的血管舒张功能无变化(P>0.05)。提示长期的阿托伐他汀治疗可改善增龄相关的内皮功能障碍。二、血管增龄性内皮功能障碍以及阿托伐他汀的干预的机制
为了进一步探讨血管增龄性的内皮功能障碍的发生机制以及阿托伐他汀的干预机制,我们观察了自然衰老组和阿托伐他汀干预组大鼠主动脉氧化应激损伤以及NO通路相关指标变化。氧化应激损伤指标：比色法测超氧化物歧化酶(Superoxide dismutase, SOD)活性和丙二醛(Malondialdehyde, MDA)含量。NO通路相关指标：Griess法检测总NO含量,以及钙依赖性的NOS活性以及非钙依赖性的NOS活性。Real-time定量PCR法测定eNOS和iNOS的mRNA含量,Western-blotting法检测NOS和iNOS的蛋白表达,免疫组化法定位检测eNOS和iNOS的表达。结果显示：(1)青年组、中年对照组和老年对照组的两两比较,随着年龄增长,SOD活性下降(P<0.05或者0.01),MDA含量升高(P<0.01),总NO含量下降(P<0.05或0.01),钙依赖性NOS活性随着年龄增长而下降(P<0.01),而非钙依赖性NOS活性随着年龄增长而上升(P<0.01)。与中年对照组比较,老年对照组的eNOS的表达显著降低(P<0.01),iNOS的表达显著升高(P<0.01), eNOS/iNOS比值显著降低(P<0.01)。虽然青年对照组eNOS表达不大于中年对照组,但是青年对照组未检测至iNOS的mRNA和蛋白的表达。这意味着eNOS/iNOS比例随着增龄过程而下降。这提示增龄导致血管氧化应激损伤和NOS系统失衡。(2)与老年对照组比较,长期阿托伐他汀干预可增加SOD含量(P<0.01),减少MDA含量(P<0.01),增加总NO含量(P<0.01),提高钙依赖性的NOS活性而降低非钙依赖性的NOS活性(P<0.01)。通过增加‘NOS的表达(P<0.01)和抑制iNOS的表达(未检测到)而恢复eNOS/iNOS比例。这提示长期阿托伐他汀干预可以减弱氧化应激损伤和恢复NOS系统的平衡。(3)免疫组化结果显示eNOS只表达在主动脉内膜(内皮细胞)上,而iNOS表达在内膜和中膜(血管平滑肌细胞)上,并且青年对照组和老年阿托伐他汀组均未检测至JiNOS的表达。这提示iNOS随着增龄而被激活,同时长期阿托伐他汀干预可以抑制iNOS的表达而恢复NOS系统的平衡。
三、SIRT1在血管增龄性内皮功能障碍及阿托伐他汀的干预的作用初探
为了探讨SIRT1在血管老化中的作用以及以及阿托伐他汀对SIRT1的影响。我们观察了各组大鼠主动脉SIRT1的变化。Real-time定量PCR法测定SIRT1mRNA含量,Western-blotting法检钡HSIRT1的蛋白定量表达,免疫组化法定位检测HSIRT1的表达。为了分析SIRT1与NOS系统的相互作用对于血管老化的影响,以及在阿托伐他汀干预中所起的作用,我们将SIRT1和NOS系统进行了相关性分析。结果显示:(1)青年对照组、中年对照组和老年对照组之间的多重比较显示,与中年对照组比较,老年对照组的SIRT1表达下降(P<0.01)。虽然青年对照组的SIRT1表达并不大于中年对照组,但是其表达高于老年对照组(P<0.01)。这提示SIRT1的表达随着衰老而降低。与其他组相比,老年阿托伐他汀组的SIRT1表达增加(P<0.01)。免疫组化显示SIRT1同时表达在血管内皮细胞和血管平滑肌细胞上。这提示长期的阿托伐他汀干预可以上调胸主动脉SIRT1的表达。(2)胸主动脉SIRT1与NOS的相关性分析显示,在所有组中,eNOS的表达与SIRT1的表达成显著正相关关系(P<0.01),相关系数在转录和翻译水平分别达到0.956和0.523。iNOS的表达与SIRT1的表达成显著负相关关系(P<0.05或者P<0.01),相关系数在转录和翻译水平分别达到-0.614和.0.872。eNOS/iNOS比例与SIRT1的表达成显著正相关关系(P<0.01),相关系数在转录和翻译水平分别达到0.987和0.905。因为iNOS仅仅在中年对照组和老年对照组被检测到,所以涉及iNOS的相关性分析只在这两组进行。尽管如此,考虑到青年对照组和老年阿托伐他汀组eNOS的相对高表达,并且该两组并未检测至iNOS的表达,所以可以合理推测该两组eNOS/iNOS比例与SIRT1的表达也是成显著正相关关系。
结论：
通过本研究,我们能够得出以下结论：
1.增龄过程中,雄性Wistar大鼠出现血管内皮功能障碍,提示衰老可以引起内皮功能障碍。衰老可通过增加细胞内氧化应激,下调eNOS,激活并上调iNOS引起NOS系统失衡,造成NO生物利用度下降,最终导致内皮功能障碍。
2.长期阿托伐他汀(5mg/kg-d)干预可改善内皮功能障碍。其机制涉及减弱氧化应激损伤,上调eNOS,抑制iNOS而恢复NOS系统平衡,提高NO生物利用度,从而改善血管内皮功能障碍。
3.增龄过程中,血管老化伴随着SIRT1表达下调,且和NOS系统的平衡密切相关。阿托伐他汀可能通过上调SIRT1而维持NOS系统平衡而改善血管内皮功能障碍。
Population aging is the decrease of the number of young population and the increase of the number of the elderly population in the total population, which causes a corresponding increase of proportion of the elderly population in the total population. Population aging is a worldwide problem from the second half phase of the20th century to the early phase of21st century appears. Currently the aging population of the world who aged60and above reached750million. By2050, the aging population is expected to reach2billion, and80%of old people will live in the developing countries. By2013, China's population aged60or older had reached202million, accounting for14.8%of the total population; By2015and2020, this proportion will reach15%and18%respectively. Typically, the ratio of the population aged65or older is more than7%, it is called " aging society ", while more than14%is called " aged society." All developed countries in the world have entered the aging society, many developing countries have entered or are about to enter an aging society. In1999, China entered the " aging society ", and the physical condition of the elderly was a disturbing problem. The demand of medical treatment, health care, nursing services and living services of old people are much more than other people. Therefore, proposing healthy aging and improving the physical condition of old people is the mission of health care providers and the society. Theoretically anti-aging treatment can alter the biochemical and molecular changes of aging, rectify the physiological changes associated with aging, reduce susceptibility to aging-related diseases. American Academy of Anti-Aging Medicine Society (A4M) declared:" The age-related disability is caused by the body's physiological dysfunction, such dysfunction can be improved by medical treatment in many cases. Human life span is expected to be extended, and the quality of life of old people is expected to be improved." Human aging can be interpreted and may be slowed down. Searching molecular targets of anti-aging and exerting corresponding intervention is the current hotspot.
Because the normal operation of the whole body depends on the blood supply of circulatory system, and the normal function of blood vessels plays an indispensable role in the normal operation of the circulatory system. Therefore, the changes of vascular function in the aging process can greatly affect overall body function. With the aging of the body, the blood vessels also aged correspondingly, which is characterized by the changes of the vascular structure and function. Age-related vascular dysfunction is closely related to endothelial dysfunction, which is characterized by progressive decrease of endothelium-dependent vasodilatation capacity of the resistance and conductivity arteries. Endothelial dysfunction is a systemic dysfunction, and is a key factor of atherosclerosis, coronary heart disease and hypertension. Therefore, improving endothelial dysfunction can reduce the morbidity and mortality of cardiovascular and cerebrovascular diseases. Although in vitro experiments have demonstrated inhibition of vascular cell senescence can improve cell dysfunction, in vivo research still needs to be further explored.
Statins, a3-hydroxy-3-methyl glutaric acid coenzyme A reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) inhibitors, not only have an effect of regulating lipid, but also can improve endothelial dysfunction, consequently preventing a range of vascular-related diseases, such as atherosclerosis, coronary heart disease and hypertension. However, previous studies mainly focused on the effects of in vitro or short-term statin intervention on acute pathologic vascular dysfunction models. The slow and slight pathological changes during the aging process needs a long time to accumulate to cause significant functional changes, so establishing a natural aging animal models is a better strategy to study the effect of statins on age-related vascular dysfunction.
Several signaling pathways have been shown to participate in vascular aging and the inhibitory effect of statins. Nitric oxide (NO) has been recognized as a key signaling molecule in the regulation of vascular function. NO is synthesized by nitric oxide synthases (NOS), which are composed of three isoforms, namely endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). The effect of NO on vascular function depends on its environment, including the sources and amounts of NO. eNOS and eNOS-derived NO are calcium-dependent and play a useful role, but decreases with aging. iNOS and iNOS-derived NO are calcium-independent and play harmful effects, but increases with aging. Under physiological conditions, eNOS-derived NO has an effect of vasodilatation, thus maintaining normal vascular function. Under pathological conditions, iNOS is activated and produces large amounts of NO. On the one hand, excessive NO interacts with reactive oxygen species (ROS) to form peroxynitrite (ONOO-), and then induces lipid peroxidation to accelerate vascular aging, consequently results in endothelial dysfunction. On the other hand, iNOS-derived NO activates arginase I to cause eNOS substrate degradation and eNOS uncoupling which consequently results in vascular dysfunction. During the aging process, iNOS of the aorta is activated or upregulated, but the exact location and source of iNOS needs more research. The effect of Statins on NOS isoforms is also controversial, especially on iNOS. Because the natural aging animal models require long-term intervention and observation, the effect of statins on endothelial dysfunction of natural aging animal model is unclear and needs further study.
SIRT1(Sirtuin-1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone, can regulate endothelial cell senescence and vascular function. SIRT1can interact with eNOS, and stimulate the expression of eNOS to inhibit vascular cell senescence and improve endothelial dysfunction. Although in vitro experiments have confirmed that atorvastatin could induce SIRT1to inhibit cell senescence, but whether atorvastatin can also inhibit vascular aging by inducing SIRT1needs further study.
Based on the above ideas and research status, we assume that vascular dysfunction will happen during aging, and that long-term atorvastatin intervention can improve vascular dysfunction by regulating oxidative stress injury and NOS system imbalance during aging. Meanwhile, based on the role of SIRT1in vascular function, we assume that SIRT1can interact with the NOS system changes to regulate vascular function and the effects of statins. Our previous studies have shown, after long-term feeding, blood vessels from Wistar rats of different ages developed a series of pathological changes, including oxidative stress injury and the downregulation of eNOS. These age-related changes suggested rat blood vessels developed aging, so the model can be used as a good animal model of natural aging. In this study, an improved animal model which is composed of male Wistar rats of different ages with long-term feeding was used as the natural aging model. This study focused on the following three aspects, which not only can answer the above hypothetical questions, but also can prove whether long-term atorvastatin improves age-related endothelial dysfunction by regulating classic NOS system and SIRT1.
Rat age-related vascular endothelial dysfunction and the effect of atorvastatin
Two currently widely accepted indicators were used to test vascular function: The vascular endothelium-dependent function test (acetylcholine-induced vasodilatation test) and vascular endothelium-independent function test (sodium nitroprusside-induced vasodilatation test). The rats were divided into4groups: young control group (2-month-old male Wistar rats, n=8), the middle-aged control group (4-month-old male Wistar rats received a sterilized diet ad libitum until12months old, n=8); the old control group (12-month-old male Wistar rats received a sterilized diet ad libitum until20months old, n=8; At the age of19months, one rat died); the old atorvastatin group (12-month-old male Wistar rats received a sterilized diet mixed with atorvastatin at a dose of5mg/kg/day until20months old, n=8). After eight months, the rat underwent thoracotomy and the thoracic aortas were extracted, and then above indicators were measured. The results showed that: Multiple comparisons between YC, MC, and OC showed that endothelium-dependent relaxation in response to acetylcholine decreased with age (P<0.05or0.01). Multiple comparisons of endothelium-independent relaxation in response to sodium nitroprusside showed no differences between YC, MC and OC. Such results suggested that aging caused rat vascular endothelial dysfunction.
In addition, the effect of long-term atorvastatin intervention on vascular function showed:Compared with OC, long-term administration of atorvastatin (OA) attenuated the reduction of endothelium-dependent relaxation (P<0.05or0.01), endothelium-independent vasodilatation did not change (P>0.05). Such results suggested that long-term atorvastatin intervention can improve age-related endothelial dysfunction.
The mechanisms of age-related vascular endothelial dysfunction and atorvastatin intervention
To further investigate the mechanisms of age-related vascular endothelial dysfunction and atorvastatin intervention, we observed aortic oxidative stress and NO pathway related changes in the natural aging groups and old atorvastatin group. Oxidative stress indicators:Superoxide dismutase (SOD) activity and Malondialdehyde (MDA) content were measured by colorimetric method. NO pathway related indicators:total NO content and NOS activity of calcium-dependent and calcium-independent were measured by Griess assay. eNOS and iNOS mRNA levels were measurd by Real-time quantitative PCR assay. The expressions of eNOS and iNOS proteins were measured by Western-blotting. The localization expression of eNOS and iNOS were detected by immunohistochemistry. The results showed that:(1) Multiple comparisons between YC, MC, and OC, SOD activity decreased (P<0.05or0.01), MDA content increased (P<0.01), total NO content decreased (P<0.05or0.01), calcium-dependent NOS activity declined with increasing age (P<0.01), but calcium-independent NOS activity increased with increasing age (P<0.01). Compared with MC, the eNOS expression level in OC decreased significantly (P<.001),the iNOS expression level increased significantly (P<0.01), and the eNOS/iNOS ratio decreased significantly (P<0.01). Although the eNOS expression level in YC was not greater than that in MC, the iNOS expression level in YC was detected by neither real-time PCR nor western blotting which suggested that the eNOS/iNOS ratio declined with age. This suggests that aging leads to vascular oxidative stress and NOS system imbalance.(2) Compared with OC group, long-term atorvastatin intervention increased SOD levels (P<0.01), reduced the content of MDA (P<0.01), increased total NO levels (P<0.01), increased calcium-dependent NOS activity and reduced calcium-independent NOS activity (P<0.01). Increasing the expression of eNOS (P<0.01) and inhibition of iNOS expression (not detected) restored eNOS/iNOS ratio. This suggests that long-term atorvastatin can reduce oxidative stress and restore NOS system imbalance.(3) Immunohistochemistry showed that eNOS only expressed in aortic intima (endothelial cells), while iNOS expressed both in the intima and media (vascular smooth muscle cells), and iNOS were not detected in YC and OA groups. This suggests that iNOS is activated with increasing age, while long-term atorvastatin intervention could inhibit the expression of iNOS and restore NOS system imbalance.
The effect of SIRT1on age-related vascular endothelial dysfunction and atorvastatin intervention
In order to investigate the role of SIRT1in vascular aging and the effects of atorvastatin on SIRT1, the changes of aorta SIRT1were observed. SIRT1mRNA level was measured by Real-time quantitative PCR assay. The expression of SIRT1protein was measured by Western-blotting. The localization expression of SIRT1was detected by immunohistochemistry. In order to analyze the effecs of the interaction of SIRT1and NOS system on vascular aging and the role of atorvastatin intervention, SIRT1and NOS system correlation was analyzed. The results showed that:(1) Compared with MC, the SIRT1expression level in OC decreased significantly (P<0.01). Although the SIRT1expression level in YC was not more than that in MC, it was more than that in OC (P<0.01). These results suggested that SIRT1expression gradually declined with age. Compared with all other groups, long-term administration of atorvastatin (OA) not only restored but also increased SIRT1expression (P<0.01). Immunohistochemistry showed that SIRT1expressed in both the endothelium and VSMCs.This suggested that atorvastatin was able to upregulate SIRT1expression.(2) Correlation analysis of thoracic aortic SIRT1and NOS showed that In all groups, eNOS expression was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.956and0.523at the transcriptional and translational levels respectively. iNOS expression was significantly negatively correlated with SIRT1expression (P<0.05or0.01); the correlation coefficients reached-0.614and-0.872at the transcriptional and translational levels respectively. The eNOS/iNOS ratio was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.987and0.905at the transcriptional and translational levels respectively. Because iNOS expression was detected only in MC and OC, correlation analysis involving iNOS expression was only conducted in these two groups. However, in view of the relatively high expression of eNOS in YC and OA, as well as no detection of iNOS in these two groups, it is reasonable to speculate that eNOS/iNOS ratio were also significantly positively correlated with SIRT1expression in these two groups.
Conclusion:
1During aging process, male Wistar rats developed vascular endothelial dysfunction, which suggested that aging can cause endothelial dysfunction. By increasing intracellular oxidative stress, downregulating eNOS and activating iNOS to cause NOS system imbalance, aging caused decreased NO bioavailability, consequently led to endothelial dysfunction.
2Long-term atorvastatin (5mg/kg· d) intervention can improve age-related endothelial dysfunction. By reducing oxidative stress, upregulating eNOS, inhibiting iNOS to restore NOS system imbalance, atorvastatin increased NO bioavailability, consequently improved endothelial dysfunction.
3During aging, vascular aging was accompanied by downregulation of SIRT1and was closely associated with NOS system imbalance. Atorvastatin maybe increase SIRT1to mantain NOS system balance and improve endothelial dysfunction.
因为全身各器官组织的正常工作有赖于循环系统的正常供血,而血管的正常功能在维持循环系统的正常运行中发挥着不可或缺的作用,所以衰老过程中血管功能的改变将极大地影响着人体整体的机能。随着机体的衰老,血管也相应地衰老,表现为血管结构和功能的改变。增龄性血管功能障碍与内皮功能障碍密切相关,表现为阻力和传导动脉的内皮依赖性血管舒张能力进行性下降。因为血管内皮功能障碍是一个系统性的功能紊乱,并且是动脉粥样硬化、冠心病和高血压病的一个关键因素,因此改善内皮功能障碍能够减少心脑血管疾病的发病率和死亡率。虽然离体实验已经证实抑制血管细胞衰老能够改善细胞功能障碍,但是在体的研究仍然需要进一步探讨。
他汀作为3-羟基-3-甲基戊二酸辅酶A还原酶(3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase)抑制剂,除具有调脂作用外,还可以改善血管内皮功能障碍,从而防治一系列的血管相关疾病,如动脉粥样硬化、冠心病和高血压等。尽管如此,之前的研究主要是离体实验,或者短期的他汀干预急性病理血管功能障碍模型。考虑到衰老过程中缓慢而微小的病理变化需要长期的累积才能造成明显的功能改变,建立一个自然衰老动物模型将能够更好地研究他汀对于增龄性血管功能障碍的影响。
几个信号通路已经被证实参与血管衰老以及他汀的抑制效应。一氧化氮(NO)被公认为调节血管功能的关键信号分子。NO由一氧化氮合成酶(Nitric oxide synthase, NOS)合成,而一氧化氮合成酶有三种同分异构体,分别为内皮型一氧化氮合成酶(endothelial Nitric oxide synthase, eNOS)、诱导型一氧化氮合成酶(inducible Nitric oxide synthase, iNOS)和神经型一氧化氮合成酶(neuronal Nitric oxide synthase, nNOS)。NO对于血管功能的影响取决于它所处的环境,包括NO的来源和数量。eNOS以及eNOS来源的NO是钙依赖性的,并发挥有益的作用,但是随着衰老而下降。iNOS以及iNOS来源的NO是非钙依赖性的,并发挥有害的作用,但是随着衰老而上升。生理情况下eNOS合成的NO使血管舒张,从而维持正常血管功能。在病理情况下,iNOS被激活而产生大量NO。一方面,过量NO与活性氧簇(Reactive oxygen species, ROS)相互作用,形成亚硝酸盐(Peroxynitrite, ONOO-),进而诱导脂质过氧化而加速血管衰老,最终导致内皮功能障碍。另一方面,iNOS来源的NO激活精氨酸酶I,使eNOS底物降解,导致eNOS的失偶联而造成血管功能障碍。衰老过程中,主动脉中的iNOS被激活或上调,但是对于iNOS的确切位置和来源依然值得研究。他汀对于NOS同分异构体的作用也存在争议,特别是对于iNOS的影响。因为自然衰老动物模型需要长期的干预和观察,他汀对于自然衰老动物模型内皮功能障碍的研究甚少,需要进一步研究。
SIRT1(Sirtuin-1)是一个烟酰胺腺嘌呤二核苷酸(NAD+)依赖的组蛋白,能够调节内皮细胞衰老和血管功能。SIRT1也能够与eNOS相互作用,并刺激eNOS的表达从而抑制血管细胞衰老和改善内皮功能障碍。虽然离体实验证实,阿托伐他汀可以通过诱导SIRT1抑制细胞衰老,但是阿托伐他汀是否可以同样通过诱导SIRT1而抑制血管老化仍值得探讨。
基于以上观点和研究现状,我们假设衰老过程中血管将出现功能障碍,阿托伐他汀的长期干预治疗可以改善血管功能障碍,并且是通过调节增龄性变化过程的氧化应激损伤以及NOS系统的失衡的结果。同时,基于SIRT1在血管功能中发挥的作用,我们假设SIRT1也能够与NOS系统相互作用,而参与血管功能的改变以及他汀的干预效应。本课题组前期研究显示,不同年龄段的Wistar大鼠通过长期饲养,血管出现一系列的病理变化,包括氧化应激损伤和eNOS的下调。这些增龄性变化提示大鼠血管发生了衰老,因此该模型可作为良好的自然衰老动物模型。本研究采用改进的动物模型,采用各个年龄段雄性Wistar大鼠长期喂养,作为自然衰老模型。本研究围绕以下三个方面进行,不但可以回答上述提出的假设问题,同时可以探讨阿托伐他汀改善内皮功能障碍的作用是否通过其经典的NOS系统,以及对于SIRT1的影响。
一、大鼠血管增龄性内皮功能障碍以及阿托伐他汀的干预作用
本研究以目前公认的反映血管功能的两项指标：内皮依赖性的血管功能检查(乙酰胆碱诱导的血管舒张能力检测)以及非内皮依赖性的血管功能检查(硝普钠诱导的血管舒张能力检测)为主要观察指标。实验分4组：青年对照组(2月龄雄性Wistar大鼠,n=8),中年对照组(4月龄雄性Wistar大鼠喂养基础饲料,饲养至12月龄,n=8),老年对照组(12月龄雄性Wistar大鼠喂养基础饲料,饲养至20月龄,n=8,饲养至19月龄时,死亡1只),阿托伐他汀干预组(12月龄雄性Wistar大鼠喂养添加阿托伐他汀(5mg/kg·d)的饲料,饲养至20月龄,n=8)。饲养8个月后,开胸取胸主动脉,测定上述指标。结果显示：青年组、中年对照组和老年对照组之间的比较发现,内皮依赖性的血管舒张功能随着年龄增长而下降(P<0.05或者0.01),非内皮依赖性的血管舒张功能随着年龄增长无变化(P>0.05)。这提示增龄导致了大鼠血管内皮功能障碍。
此外,长期的阿托伐他汀干预对血管功能影响显示：与老年对照组相比,长期的阿托伐他汀干预后,内皮依赖性舒张能力得到了提高(P<0.05或者0.01),非内皮依赖性的血管舒张功能无变化(P>0.05)。提示长期的阿托伐他汀治疗可改善增龄相关的内皮功能障碍。二、血管增龄性内皮功能障碍以及阿托伐他汀的干预的机制
为了进一步探讨血管增龄性的内皮功能障碍的发生机制以及阿托伐他汀的干预机制,我们观察了自然衰老组和阿托伐他汀干预组大鼠主动脉氧化应激损伤以及NO通路相关指标变化。氧化应激损伤指标：比色法测超氧化物歧化酶(Superoxide dismutase, SOD)活性和丙二醛(Malondialdehyde, MDA)含量。NO通路相关指标：Griess法检测总NO含量,以及钙依赖性的NOS活性以及非钙依赖性的NOS活性。Real-time定量PCR法测定eNOS和iNOS的mRNA含量,Western-blotting法检测NOS和iNOS的蛋白表达,免疫组化法定位检测eNOS和iNOS的表达。结果显示：(1)青年组、中年对照组和老年对照组的两两比较,随着年龄增长,SOD活性下降(P<0.05或者0.01),MDA含量升高(P<0.01),总NO含量下降(P<0.05或0.01),钙依赖性NOS活性随着年龄增长而下降(P<0.01),而非钙依赖性NOS活性随着年龄增长而上升(P<0.01)。与中年对照组比较,老年对照组的eNOS的表达显著降低(P<0.01),iNOS的表达显著升高(P<0.01), eNOS/iNOS比值显著降低(P<0.01)。虽然青年对照组eNOS表达不大于中年对照组,但是青年对照组未检测至iNOS的mRNA和蛋白的表达。这意味着eNOS/iNOS比例随着增龄过程而下降。这提示增龄导致血管氧化应激损伤和NOS系统失衡。(2)与老年对照组比较,长期阿托伐他汀干预可增加SOD含量(P<0.01),减少MDA含量(P<0.01),增加总NO含量(P<0.01),提高钙依赖性的NOS活性而降低非钙依赖性的NOS活性(P<0.01)。通过增加‘NOS的表达(P<0.01)和抑制iNOS的表达(未检测到)而恢复eNOS/iNOS比例。这提示长期阿托伐他汀干预可以减弱氧化应激损伤和恢复NOS系统的平衡。(3)免疫组化结果显示eNOS只表达在主动脉内膜(内皮细胞)上,而iNOS表达在内膜和中膜(血管平滑肌细胞)上,并且青年对照组和老年阿托伐他汀组均未检测至JiNOS的表达。这提示iNOS随着增龄而被激活,同时长期阿托伐他汀干预可以抑制iNOS的表达而恢复NOS系统的平衡。
三、SIRT1在血管增龄性内皮功能障碍及阿托伐他汀的干预的作用初探
为了探讨SIRT1在血管老化中的作用以及以及阿托伐他汀对SIRT1的影响。我们观察了各组大鼠主动脉SIRT1的变化。Real-time定量PCR法测定SIRT1mRNA含量,Western-blotting法检钡HSIRT1的蛋白定量表达,免疫组化法定位检测HSIRT1的表达。为了分析SIRT1与NOS系统的相互作用对于血管老化的影响,以及在阿托伐他汀干预中所起的作用,我们将SIRT1和NOS系统进行了相关性分析。结果显示:(1)青年对照组、中年对照组和老年对照组之间的多重比较显示,与中年对照组比较,老年对照组的SIRT1表达下降(P<0.01)。虽然青年对照组的SIRT1表达并不大于中年对照组,但是其表达高于老年对照组(P<0.01)。这提示SIRT1的表达随着衰老而降低。与其他组相比,老年阿托伐他汀组的SIRT1表达增加(P<0.01)。免疫组化显示SIRT1同时表达在血管内皮细胞和血管平滑肌细胞上。这提示长期的阿托伐他汀干预可以上调胸主动脉SIRT1的表达。(2)胸主动脉SIRT1与NOS的相关性分析显示,在所有组中,eNOS的表达与SIRT1的表达成显著正相关关系(P<0.01),相关系数在转录和翻译水平分别达到0.956和0.523。iNOS的表达与SIRT1的表达成显著负相关关系(P<0.05或者P<0.01),相关系数在转录和翻译水平分别达到-0.614和.0.872。eNOS/iNOS比例与SIRT1的表达成显著正相关关系(P<0.01),相关系数在转录和翻译水平分别达到0.987和0.905。因为iNOS仅仅在中年对照组和老年对照组被检测到,所以涉及iNOS的相关性分析只在这两组进行。尽管如此,考虑到青年对照组和老年阿托伐他汀组eNOS的相对高表达,并且该两组并未检测至iNOS的表达,所以可以合理推测该两组eNOS/iNOS比例与SIRT1的表达也是成显著正相关关系。
结论：
通过本研究,我们能够得出以下结论：
1.增龄过程中,雄性Wistar大鼠出现血管内皮功能障碍,提示衰老可以引起内皮功能障碍。衰老可通过增加细胞内氧化应激,下调eNOS,激活并上调iNOS引起NOS系统失衡,造成NO生物利用度下降,最终导致内皮功能障碍。
2.长期阿托伐他汀(5mg/kg-d)干预可改善内皮功能障碍。其机制涉及减弱氧化应激损伤,上调eNOS,抑制iNOS而恢复NOS系统平衡,提高NO生物利用度,从而改善血管内皮功能障碍。
3.增龄过程中,血管老化伴随着SIRT1表达下调,且和NOS系统的平衡密切相关。阿托伐他汀可能通过上调SIRT1而维持NOS系统平衡而改善血管内皮功能障碍。
Population aging is the decrease of the number of young population and the increase of the number of the elderly population in the total population, which causes a corresponding increase of proportion of the elderly population in the total population. Population aging is a worldwide problem from the second half phase of the20th century to the early phase of21st century appears. Currently the aging population of the world who aged60and above reached750million. By2050, the aging population is expected to reach2billion, and80%of old people will live in the developing countries. By2013, China's population aged60or older had reached202million, accounting for14.8%of the total population; By2015and2020, this proportion will reach15%and18%respectively. Typically, the ratio of the population aged65or older is more than7%, it is called " aging society ", while more than14%is called " aged society." All developed countries in the world have entered the aging society, many developing countries have entered or are about to enter an aging society. In1999, China entered the " aging society ", and the physical condition of the elderly was a disturbing problem. The demand of medical treatment, health care, nursing services and living services of old people are much more than other people. Therefore, proposing healthy aging and improving the physical condition of old people is the mission of health care providers and the society. Theoretically anti-aging treatment can alter the biochemical and molecular changes of aging, rectify the physiological changes associated with aging, reduce susceptibility to aging-related diseases. American Academy of Anti-Aging Medicine Society (A4M) declared:" The age-related disability is caused by the body's physiological dysfunction, such dysfunction can be improved by medical treatment in many cases. Human life span is expected to be extended, and the quality of life of old people is expected to be improved." Human aging can be interpreted and may be slowed down. Searching molecular targets of anti-aging and exerting corresponding intervention is the current hotspot.
Because the normal operation of the whole body depends on the blood supply of circulatory system, and the normal function of blood vessels plays an indispensable role in the normal operation of the circulatory system. Therefore, the changes of vascular function in the aging process can greatly affect overall body function. With the aging of the body, the blood vessels also aged correspondingly, which is characterized by the changes of the vascular structure and function. Age-related vascular dysfunction is closely related to endothelial dysfunction, which is characterized by progressive decrease of endothelium-dependent vasodilatation capacity of the resistance and conductivity arteries. Endothelial dysfunction is a systemic dysfunction, and is a key factor of atherosclerosis, coronary heart disease and hypertension. Therefore, improving endothelial dysfunction can reduce the morbidity and mortality of cardiovascular and cerebrovascular diseases. Although in vitro experiments have demonstrated inhibition of vascular cell senescence can improve cell dysfunction, in vivo research still needs to be further explored.
Statins, a3-hydroxy-3-methyl glutaric acid coenzyme A reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) inhibitors, not only have an effect of regulating lipid, but also can improve endothelial dysfunction, consequently preventing a range of vascular-related diseases, such as atherosclerosis, coronary heart disease and hypertension. However, previous studies mainly focused on the effects of in vitro or short-term statin intervention on acute pathologic vascular dysfunction models. The slow and slight pathological changes during the aging process needs a long time to accumulate to cause significant functional changes, so establishing a natural aging animal models is a better strategy to study the effect of statins on age-related vascular dysfunction.
Several signaling pathways have been shown to participate in vascular aging and the inhibitory effect of statins. Nitric oxide (NO) has been recognized as a key signaling molecule in the regulation of vascular function. NO is synthesized by nitric oxide synthases (NOS), which are composed of three isoforms, namely endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). The effect of NO on vascular function depends on its environment, including the sources and amounts of NO. eNOS and eNOS-derived NO are calcium-dependent and play a useful role, but decreases with aging. iNOS and iNOS-derived NO are calcium-independent and play harmful effects, but increases with aging. Under physiological conditions, eNOS-derived NO has an effect of vasodilatation, thus maintaining normal vascular function. Under pathological conditions, iNOS is activated and produces large amounts of NO. On the one hand, excessive NO interacts with reactive oxygen species (ROS) to form peroxynitrite (ONOO-), and then induces lipid peroxidation to accelerate vascular aging, consequently results in endothelial dysfunction. On the other hand, iNOS-derived NO activates arginase I to cause eNOS substrate degradation and eNOS uncoupling which consequently results in vascular dysfunction. During the aging process, iNOS of the aorta is activated or upregulated, but the exact location and source of iNOS needs more research. The effect of Statins on NOS isoforms is also controversial, especially on iNOS. Because the natural aging animal models require long-term intervention and observation, the effect of statins on endothelial dysfunction of natural aging animal model is unclear and needs further study.
SIRT1(Sirtuin-1), a nicotinamide adenine dinucleotide (NAD+)-dependent histone, can regulate endothelial cell senescence and vascular function. SIRT1can interact with eNOS, and stimulate the expression of eNOS to inhibit vascular cell senescence and improve endothelial dysfunction. Although in vitro experiments have confirmed that atorvastatin could induce SIRT1to inhibit cell senescence, but whether atorvastatin can also inhibit vascular aging by inducing SIRT1needs further study.
Based on the above ideas and research status, we assume that vascular dysfunction will happen during aging, and that long-term atorvastatin intervention can improve vascular dysfunction by regulating oxidative stress injury and NOS system imbalance during aging. Meanwhile, based on the role of SIRT1in vascular function, we assume that SIRT1can interact with the NOS system changes to regulate vascular function and the effects of statins. Our previous studies have shown, after long-term feeding, blood vessels from Wistar rats of different ages developed a series of pathological changes, including oxidative stress injury and the downregulation of eNOS. These age-related changes suggested rat blood vessels developed aging, so the model can be used as a good animal model of natural aging. In this study, an improved animal model which is composed of male Wistar rats of different ages with long-term feeding was used as the natural aging model. This study focused on the following three aspects, which not only can answer the above hypothetical questions, but also can prove whether long-term atorvastatin improves age-related endothelial dysfunction by regulating classic NOS system and SIRT1.
Rat age-related vascular endothelial dysfunction and the effect of atorvastatin
Two currently widely accepted indicators were used to test vascular function: The vascular endothelium-dependent function test (acetylcholine-induced vasodilatation test) and vascular endothelium-independent function test (sodium nitroprusside-induced vasodilatation test). The rats were divided into4groups: young control group (2-month-old male Wistar rats, n=8), the middle-aged control group (4-month-old male Wistar rats received a sterilized diet ad libitum until12months old, n=8); the old control group (12-month-old male Wistar rats received a sterilized diet ad libitum until20months old, n=8; At the age of19months, one rat died); the old atorvastatin group (12-month-old male Wistar rats received a sterilized diet mixed with atorvastatin at a dose of5mg/kg/day until20months old, n=8). After eight months, the rat underwent thoracotomy and the thoracic aortas were extracted, and then above indicators were measured. The results showed that: Multiple comparisons between YC, MC, and OC showed that endothelium-dependent relaxation in response to acetylcholine decreased with age (P<0.05or0.01). Multiple comparisons of endothelium-independent relaxation in response to sodium nitroprusside showed no differences between YC, MC and OC. Such results suggested that aging caused rat vascular endothelial dysfunction.
In addition, the effect of long-term atorvastatin intervention on vascular function showed:Compared with OC, long-term administration of atorvastatin (OA) attenuated the reduction of endothelium-dependent relaxation (P<0.05or0.01), endothelium-independent vasodilatation did not change (P>0.05). Such results suggested that long-term atorvastatin intervention can improve age-related endothelial dysfunction.
The mechanisms of age-related vascular endothelial dysfunction and atorvastatin intervention
To further investigate the mechanisms of age-related vascular endothelial dysfunction and atorvastatin intervention, we observed aortic oxidative stress and NO pathway related changes in the natural aging groups and old atorvastatin group. Oxidative stress indicators:Superoxide dismutase (SOD) activity and Malondialdehyde (MDA) content were measured by colorimetric method. NO pathway related indicators:total NO content and NOS activity of calcium-dependent and calcium-independent were measured by Griess assay. eNOS and iNOS mRNA levels were measurd by Real-time quantitative PCR assay. The expressions of eNOS and iNOS proteins were measured by Western-blotting. The localization expression of eNOS and iNOS were detected by immunohistochemistry. The results showed that:(1) Multiple comparisons between YC, MC, and OC, SOD activity decreased (P<0.05or0.01), MDA content increased (P<0.01), total NO content decreased (P<0.05or0.01), calcium-dependent NOS activity declined with increasing age (P<0.01), but calcium-independent NOS activity increased with increasing age (P<0.01). Compared with MC, the eNOS expression level in OC decreased significantly (P<.001),the iNOS expression level increased significantly (P<0.01), and the eNOS/iNOS ratio decreased significantly (P<0.01). Although the eNOS expression level in YC was not greater than that in MC, the iNOS expression level in YC was detected by neither real-time PCR nor western blotting which suggested that the eNOS/iNOS ratio declined with age. This suggests that aging leads to vascular oxidative stress and NOS system imbalance.(2) Compared with OC group, long-term atorvastatin intervention increased SOD levels (P<0.01), reduced the content of MDA (P<0.01), increased total NO levels (P<0.01), increased calcium-dependent NOS activity and reduced calcium-independent NOS activity (P<0.01). Increasing the expression of eNOS (P<0.01) and inhibition of iNOS expression (not detected) restored eNOS/iNOS ratio. This suggests that long-term atorvastatin can reduce oxidative stress and restore NOS system imbalance.(3) Immunohistochemistry showed that eNOS only expressed in aortic intima (endothelial cells), while iNOS expressed both in the intima and media (vascular smooth muscle cells), and iNOS were not detected in YC and OA groups. This suggests that iNOS is activated with increasing age, while long-term atorvastatin intervention could inhibit the expression of iNOS and restore NOS system imbalance.
The effect of SIRT1on age-related vascular endothelial dysfunction and atorvastatin intervention
In order to investigate the role of SIRT1in vascular aging and the effects of atorvastatin on SIRT1, the changes of aorta SIRT1were observed. SIRT1mRNA level was measured by Real-time quantitative PCR assay. The expression of SIRT1protein was measured by Western-blotting. The localization expression of SIRT1was detected by immunohistochemistry. In order to analyze the effecs of the interaction of SIRT1and NOS system on vascular aging and the role of atorvastatin intervention, SIRT1and NOS system correlation was analyzed. The results showed that:(1) Compared with MC, the SIRT1expression level in OC decreased significantly (P<0.01). Although the SIRT1expression level in YC was not more than that in MC, it was more than that in OC (P<0.01). These results suggested that SIRT1expression gradually declined with age. Compared with all other groups, long-term administration of atorvastatin (OA) not only restored but also increased SIRT1expression (P<0.01). Immunohistochemistry showed that SIRT1expressed in both the endothelium and VSMCs.This suggested that atorvastatin was able to upregulate SIRT1expression.(2) Correlation analysis of thoracic aortic SIRT1and NOS showed that In all groups, eNOS expression was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.956and0.523at the transcriptional and translational levels respectively. iNOS expression was significantly negatively correlated with SIRT1expression (P<0.05or0.01); the correlation coefficients reached-0.614and-0.872at the transcriptional and translational levels respectively. The eNOS/iNOS ratio was significantly positively correlated with SIRT1expression (P<0.01); the correlation coefficients reached0.987and0.905at the transcriptional and translational levels respectively. Because iNOS expression was detected only in MC and OC, correlation analysis involving iNOS expression was only conducted in these two groups. However, in view of the relatively high expression of eNOS in YC and OA, as well as no detection of iNOS in these two groups, it is reasonable to speculate that eNOS/iNOS ratio were also significantly positively correlated with SIRT1expression in these two groups.
Conclusion:
1During aging process, male Wistar rats developed vascular endothelial dysfunction, which suggested that aging can cause endothelial dysfunction. By increasing intracellular oxidative stress, downregulating eNOS and activating iNOS to cause NOS system imbalance, aging caused decreased NO bioavailability, consequently led to endothelial dysfunction.
2Long-term atorvastatin (5mg/kg· d) intervention can improve age-related endothelial dysfunction. By reducing oxidative stress, upregulating eNOS, inhibiting iNOS to restore NOS system imbalance, atorvastatin increased NO bioavailability, consequently improved endothelial dysfunction.
3During aging, vascular aging was accompanied by downregulation of SIRT1and was closely associated with NOS system imbalance. Atorvastatin maybe increase SIRT1to mantain NOS system balance and improve endothelial dysfunction.
引文
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[6]Sanz A, R K a Stefanatos. The mitochondrial free radical theory of aging:a critical view. Current aging science,2008,1:10-21.
[7]Yildiz O. Vascular Smooth Muscle and Endothelial Functions in Aging. Annals of the New York Academy of Sciences,2007,1100:353-360.
[8]Toda N. Age-related changes in endothelial function and blood flow regulation. Pharmacology & Therapeutics,2012,133:159-176.
[9]Herrera M D, C Mingorance, R Rodriguez-Rodriguez, et al. Endothelial dysfunction and aging:An update. Ageing Research Reviews,2010,9:142-152.
[10]Taddei S V A, Mattei P, Ghiadoni L, Gennari A, Fasolo CB, Sudano I, Salvetti A. Aging and Endothelial Function in Normotensive Subjects and Patients With Essential Hypertension. Circulation,1995,91:1981-1987.
[11]Al-Shaer M H, N E Choueiri, M L G Correia, et al. Effects of aging and atherosclerosis on endothelial and vascular smooth muscle function in humans. International Journal of Cardiology,2006,109:201-206.
[12]Minamino T. Vascular cell senescence and vascular aging. Journal of Molecular and Cellular Cardiology,2004,36:175-183.
[13]Wenzel P, S Schuhmacher, J Kienhofer, et al. Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction. Cardiovascular Research,2008,80:280-289.
[14]Lee M Y K, Y Wang, P M Vanhoutte. Senescence of Cultured Porcine Coronary Arterial Endothelial Cells Is Associated with Accelerated Oxidative Stress and Activation of NFkB. Journal of Vascular Research,2010,47:287-298.
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[16]van der Loo B, R Labugger, J N Skepper, et al. Enhanced peroxynitrite formation is associated with vascular aging. The Journal of experimental medicine,2000,192: 1731-1744.
[17]Forstermann U, W C Sessa. Nitric oxide synthases:regulation and function. European Heart Journal,2011,33:829-837.
[18]Juni R P, H J Duckers, P M Vanhoutte, et al. Oxidative Stress and Pathological Changes After Coronary Artery Interventions. Journal of the American College of Cardiology,2013, 61:1471-1481.
[19]Chou T C, M H Yen, C Y Li, et al. Alterations of Nitric Oxide Synthase Expression With Aging and Hypertension in Rats. Hypertension,1998,31:643-648.
[20]Csiszar A. Aging-Induced Phenotypic Changes and Oxidative Stress Impair Coronary Arteriolar Function. Circulation Research,2002,90:1159-1166.
[21]Zanetti M, G G Cappellari, I Burekovic, et al. Caloric restriction improves endothelial dysfunction during vascular aging:Effects on nitric oxide synthase isoforms and oxidative stress in rat aorta. Experimental Gerontology,2010,45:848-855.
[22]Cernadas M R, L S de Miguel, M Garcia-Duran, et al. Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats. Circulation Research,1998,83:279-286.
[23]Goettsch W, T Lattmann, K Amann, et al. Increased Expression of Endothelin-1 and Inducible Nitric Oxide Synthase Isoform II in Aging Arteries in Vivo:Implications for Atherosclerosis. Biochemical and Biophysical Research Communications,2001,280: 908-913.
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[26]Finkel T, C-X Deng, R Mostoslavsky. Recent progress in the biology and physiology of sirtuins. Nature,2009,460:587-591.
[27]Yang Z M X. The vascular SIRTainty. Aging (Albany NY).2010,2:331-332.
[28]Nisoli E. Calorie Restriction Promotes Mitochondrial Biogenesis by Inducing the Expression of eNOS. Science,2005,310:314-317.
[29]Mattagajasingh I, C S Kim, A Naqvi, et al. SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. Proceedings of the National Academy of Sciences,2007,104:14855-14860.
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